1. Field of the Invention
The present invention relates to a method for increasing chaperone activity by irradiating peroxiredoxin proteins.
2. Description of the Related Art
Reactive oxygen species (ROS) is generated during aerobic metabolism or when a living body is exposed on a variety of stress conditions (Finkel T., Curr. Opin. Cell Biol. 15: 247-254, 2003). Such ROS causes serious damages such as oxidative functional impairments or serious structural changes of biological macromolecules (proteins, lipids, nucleic acids, etc.), which can be a cause of various diseases (Neumann et al., Nature, 424: 561-565, 2003). All the aerobic organisms have various forms of molecular chaperones, for example anti-oxidative proteins and heat-shock proteins, in order to protect themselves from protein denaturation and aggregation induced by such protein denaturation mediated by oxidative stress or ROS.
Peroxiredoxin (Prx) has been identified in most eukaryotic cells and prokaryotic cells (Chae et al., J. Biol. Chem., 269: 27670-27678, 1994). Even though peroxiredoxin does not show high homology in total amino acid sequences with the proteins having thioredoxin structure, it is still classified as thioredoxin family (Schroder et al., Protein Sci., 7: 2465-2468, 1998). Prx proteins are divided into two groups according to the number of preserved cysteine (Cys) residues, which are one cysteine Prx (1-Cys Prx) and two cysteine Prx (2-Cys Prx). More particularly, there are 5 groups of Prx proteins according to the number of well-preserved cysteine residues and structural and catalytic characteristics, which are 1-Cys Prx, 2-Cys Prx, type II Prx (Prx II), Prx Q, and GPxs. Among them, 2-Cys Prx has been most studied. It has been known that 2-Cys Prx in Arabidopsis thaliana is functioning to protect chloroplast proteins on the surface of stroma of thylakoid membrane of chloroplast from oxidative damage (Baier et al., Plant Phsiol., 199: 1407-1414, 1999). To protect chloroplast from stress induced by ROS, the cysteine residue in 2-Cys Prx active cite is oxidized into sulfenic- or sulfinic form, and the sulfenic form is deoxidized by thioredoxin-h (Trx-h), thioredoxin-f (Trx-f) and thioredoxin-m (Trx-m) (Motohashi et al., Proc. Natl. Acad. Sci., 98: 11224-11229, 2001; Balmer et al., Proc. Natl. Acad. Sci., 100: 370-375, 2003) while the sulfinic form is deoxidized by sulphiredoxin and sestrin (Beteau et al., Nature, 425: 980-984). Prx regulates peroxide mediated signal transduction, according to previous reports. In addition, Prx has many functions involved in cell proliferation, differentiation, immune response, growth regulation, cancer cell development, apoptosis, and many other unidentified functions as well (Neumann et al., Nature, 424: 561-565, 2003; Hirotsu et al., Proc. Natl. Acad. Sci. USA, 96: 12333-12338, 1999).
2-Cys Prx is known to be expressed in various cancers and neurodegenerative diseases such as Alzheimer disease, Pick disease and Down syndrome (Noh, D. Y. et al, Anticancer Res, 2001; Yanagawa, T. et al, Cancer Lett, 1999; Kinnula, V. L. et al, J. Pathol., 2002; Chang, W. J. et al, Biochem. Biophys. Res. Commun., 2001; Multhaup, G. et al, Biochem. Pharmacol, 1997; Krapfenbauer, K. et al, Brain Res., 2003). It is presumed that such expression of 2-Cys Prx is to protect cells from oxidative stress under the cancer and other degenerative disease conditions.
Peroxidase activity is continued by the following cycle: NADPH is converted to NADP+, during which H+ is delivered to thioredoxin reductase (TR); then TR delivers H+ to thioredoxin (Trx) and is oxidized; Trx deoxidized by receiving H+ delivers H+ to Prx, during which Trx is oxidized; and Prx deoxidized by receiving H+ decomposes H2O2 into O2+H2O, and this cycle is repeated with the consumption of NADPH (seen FIG. 21).
Chaperone is the protein involved in protein folding. For example, once protein gets stress like heat shock, the original tertiary structure of the protein is denatured, indicating the protein loses its function as a protein. Chaperone protein recognizes the denatured protein and then helps it be folded again.
Molecular chaperone activity is largely divided into holdase activity and foldase activity. Holdase activity is working in the following processes: Once a protein is denatured by the exposure on stress (oxidative stress or heat shock stress), some hydrophobic amino acid residues are exposed and denatured protein fragments are aggregated irregularly to make aggregates. These aggregates are decomposed by protease and at this time chaperone protein (SHSPs, DnaJ) is conjugated to some of the denatured hydrophobic amino acids to inhibit the aggregation and thus to make the protein come back to the original tertiary structure (see FIG. 22).
In the meantime, foldase activity is working in the following processes; once a new protein is synthesized by ribosomes using mRNA as a template, protein folding is induced to allow the protein to have its original tertiary structure. At this time, chaperone protein (GroEL/ES, DnaK/J/E) is conjugated to the newly extended amino acid chain to form the authentic tertiary structure (see FIG. 23).
It has been well-known that 2-Cys Prx protein has double enzyme activities of peroxidase and chaperone protein. It is also known fact that additional cysteine, in addition to the above two cysteines, affects structural change of Prx protein. In particular, Prx PP1084 protein identified from Pseudomonas putida (KT2440) by the present inventors is a kind of 2-Cys Prx having double enzyme activities. The protein has strong chaperone activity and forms comparatively high molecular structure. There is an additional cysteine between the two active cysteines and the structural change caused by that cysteine affects the strong chaperone activity.
The present inventors tried to increase chaperone activity of peroxiredoxin proteins (2-Cys and 3-Cys). As a result, the inventors confirmed that Prx protein was depolymerized, dityrosine-bond was increased, beta-sheet and random coil of 2-Cys Prx were increased, alpha helix and turn structure were decreased, and secondary structure was not observed in 3-Cys Prx protein after gamma ray irradiation. The above confirmation supported the new prospect provided by the present inventors on the structural change of a protein in relation to chaperone activity increase. The structural change of Prx induces the increase of hydrophobicity involved in chaperone activity. The present inventor completed this invention by confirming more specifically that chaperone activity of Prx protein was at least three times increased by irradiation with 15˜30 kGy of gamma ray, compared with the non-irradiated group, at which chaperone activity was optimized.